RhCMV vectors based on expression of UL36, UL128, UL130, and the Pentameric Complex (PC).

<p>RhCMV vectors based on expression of UL36, UL128, UL130, and the Pentameric Complex (PC).</p

Gene map of laboratory strains of RhCMV.

<p>Gene map shows UL36, UL128, and UL130,genes as expressed by different laboratory strains of RhCMV. Each box represents a distinct exon.</p

RhCMV 68–1.2 infects MCM.

<p>A) Study timeline showing RhCMV challenges and urine collecting in MCM. B) <i>Ex vivo</i> T cell responses in blood following RhCMV challenge. Measured as the frequency of IFNγ+TNFα+ T cells following stimulation with peptides spanning SIV protein ORFs. Corrected for frequency within memory T cell compartment. C) Absolute CD8+ cell counts in peripheral blood from four MCM following administration of CD8-depleting antibody. D) <i>Ex vivo</i> CD4+ T cell responses in BAL following RhCMV challenge. E) Western blots show shedding of RhCMV/SIV 68–1.2 vectors, but not RhCMV/SIV 68–1 vectors, in the urine of immunocompromised MCM.</p

RhCMV 68–1.2 infects immunocompetent MCM.

<p>A) <i>Ex vivo</i> T cell responses in blood following RhCMV challenge. Measured as the frequency of IFNγ+TNFα+ T cells following stimulation with peptides spanning SIV protein ORFs. Corrected for frequency within memory T cell compartment. B) <i>Ex vivo</i> CD4+ T cell responses in BAL following RhCMV challenge. C) Western blots show shedding of RhCMV/SIV 68–1.2 vectors, but not RhCMV/SIV 68–1 vectors, in the urine of fully immunocompetent MCM.</p

RhCMV replicates on primary MCM fibroblasts <i>in vitro</i> and compromises MHC-I presentation of SIV transgene peptides.

<p>A) Multi-step growth curves reveal the ability of RhCMV 68–1 and RhCMV 68–1.2 to replicate on primary RM and MCM fibroblasts (PFU = plaque forming units). B) ELISpot shows IFNγ secretion by SIV-specific CD8+ T cell lines derived from RM and MCM following stimulation with RhCMV/SIV vector-infected, MHC-matched primary fibroblasts (compared to IFNγ secretion following stimulation with peptide-pulsed primary fibroblasts).</p

Nucleotide sequence variation across RhCMV and CyCMV.

<p>A) Overall nucleotide diversity within either RhCMV isolates, CyCMV isolates, or MCM CyCMV isolates. B) Graphs indicate the nucleotide diversity across the genome, calculated using overlapping 100 bp sliding windows, incrementing by 50 bp per window. C) Amino acid alignments showing changes in UL128, UL130 and UL131a for each strain when compared to the consensus sequence. D) Amino acid alignments showing changes in gB, or E) gO and gH for each strain when compared against the consensus sequence. Colored dashes represent amino acid differences from the CMV consensus sequence.</p

Cross-Species Rhesus Cytomegalovirus Infection of Cynomolgus Macaques

<div><p>Cytomegaloviruses (CMV) are highly species-specific due to millennia of co-evolution and adaptation to their host, with no successful experimental cross-species infection in primates reported to date. Accordingly, full genome phylogenetic analysis of multiple new CMV field isolates derived from two closely related nonhuman primate species, Indian-origin rhesus macaques (RM) and Mauritian-origin cynomolgus macaques (MCM), revealed distinct and tight lineage clustering according to the species of origin, with MCM CMV isolates mirroring the limited genetic diversity of their primate host that underwent a population bottleneck 400 years ago. Despite the ability of Rhesus CMV (RhCMV) laboratory strain 68–1 to replicate efficiently in MCM fibroblasts and potently inhibit antigen presentation to MCM T cells <i>in vitro</i>, RhCMV 68–1 failed to productively infect MCM <i>in vivo</i>, even in the absence of host CD8+ T and NK cells. In contrast, RhCMV clone 68–1.2, genetically repaired to express the homologues of the HCMV anti-apoptosis gene UL36 and epithelial cell tropism genes UL128 and UL130 absent in 68–1, efficiently infected MCM as evidenced by the induction of transgene-specific T cells and virus shedding. Recombinant variants of RhCMV 68–1 and 68–1.2 revealed that expression of either UL36 or UL128 together with UL130 enabled productive MCM infection, indicating that multiple layers of cross-species restriction operate even between closely related hosts. Cumulatively, these results implicate cell tropism and evasion of apoptosis as critical determinants of CMV transmission across primate species barriers, and extend the macaque model of human CMV infection and immunology to MCM, a nonhuman primate species with uniquely simplified host immunogenetics.</p></div

Coding potential of RhCMV and CyCMV strains.

<p>Full genomes of our viral isolates were assembled and aligned to each other to compare the predicted viral coding regions. Start and stop codons were determined by comparison of all published RhCMV and CyCMV genomes and the predicted full genome coding content of RhCMV [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006014#ppat.1006014.ref011" target="_blank">11</a>] was used to determine maximal low passage viral coding potential. Hypothetical full length strain 68–1 with all lab adaptations repaired was included as a theoretical full length isolate [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006014#ppat.1006014.ref025" target="_blank">25</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006014#ppat.1006014.ref029" target="_blank">29</a>]. ORFs in our isolates showing frame shift mutations or point mutations leading to the usage of a premature stop codons compared to the majority predicted stop codon leading to shortened ORFs are indicated in blue, whereas ORF containing frameshift leading to elongations compared to the predicted majority sequence are indicated in green. ORFs that are fully or mostly missing in our isolates compared to and the predicted full genome coding content of RhCMV are highlighted in red.</p

TIGIT expressing CD8⁺ T cells have impaired cytokine responses.

<p>Representative flow cytometry plots gated on CD8⁺ T cells showing (A) TIGIT or (C) PD-1 expression against Ki-67 from a chronically HIV-infected individual. Compiled data of Ki-67⁺ CD8⁺ T cell frequency (%) separated into (B) TIGIT⁺ and TIGIT^- or (D) PD-1⁺ and PD-1^- (<i>n</i> = 20). P values were calculated by Wilcoxon matched-pairs signed ranked test. <i>Ex vivo</i> PBMCs from chronically HIV-infected individuals were stimulated with HIV Gag peptide pool and assessed for cytokine production. (E) Representative flow cytometry plots gated on CD8⁺ T cells showing TIGIT expression and either IFN-γ, IL-2, or TNF-α content after no stimulation, stimulation with an HIV-1 Gag peptide pool, or a positive control stimulation with anti-CD3 + anti-CD28 Dynabeads. (F) Compiled data of IFN-γ, IL-2, or TNF-α CD8⁺ T cell frequency (%) from TIGIT⁺ or TIGIT^- CD8⁺ T cell compartments after HIV-1 Gag peptide pool stimulation (sample group includes; AS <i>n</i> = 4, EC <i>n</i> = 3, NC <i>n</i> = 3). P values were calculated by Wilcoxon matched-pairs signed ranked test. (G) Compiled data of TIGIT and PD-1 expression on HIV-1 Gag responding cells (sample group includes; AS <i>n</i> = 4, EC <i>n</i> = 3, NC <i>n</i> = 3). P values were calculated with repeated-measures one-way ANOVA, followed by Tukey’s multiple comparisons test (*p < 0.05; **p < 0.01; ***p < 0.001). (H) Representative flow cytometry plots of intracellular perforin and granzyme B from CD8⁺ T cells expressing or not expressing TIGIT. (I) Compiled frequency (%) of intracellular perforin⁺granzyme B⁺ content from TIGIT⁺ or TIGIT^- CD8⁺ T cell compartments (AS; <i>n</i> = 12). P values were calculated by Wilcoxon matched-pairs signed ranked test. (J) Representative flow cytometry plots gated on CD8⁺ T cells showing TIGIT and CD107a expression from TIGIT isotype control, no stimulation, HIV-1 Gag peptide pool, positive control stimulation with anti-CD3 + anti-CD28 Dynabeads. Compiled data of background corrected CD107a after (K) HIV-1 Gag peptide pool (L) anti-CD3 + anti-CD28 Dynabead stimulation in TIGIT⁺ or TIGIT^- CD8⁺ T cell compartments (AS; <i>n</i> = 10). P values were calculated by Wilcoxon matched-pairs signed ranked test.</p

Common γ-chain cytokines regulate TIGIT expression on CD8⁺ T cells.

<p><i>Ex vivo</i> PBMCs from chronically HIV-1 infected individuals were stimulated with HIV-1 Gag peptide pool for 12 hours. (A) Representative flow cytometry plot gated on CD8⁺ T cells showing HIV-1 Gag pentamer with no stimulation (top panel) or HIV-1 Gag stimulation (bottom panel). (B) Representative flow cytometry plot of TIGIT expression on Penta+ and Penta- cells with no stimulation or HIV-1 Gag stimulation. (C) Graph shows compiled frequency (%) of TIGIT on Penta+ cells with no stimulation and HIV-1 Gag stimulation (<i>n</i> = 9). P values calculated with Wilcoxon matched-pairs signed-rank test. (D) Representative flow cytometry histograms gated on CD8⁺ T cells overlaid with TIGIT expression frequency before and after cytokine stimulation. Dashed line indicates TIGIT isotype control, shaded histogram indicates TIGIT expression with no stimulation, and the solid line indicates TIGIT expression with cytokine stimulation after six days. Compiled data of TIGIT frequency (%) on CD8⁺ T cells (E) HIV-Infected participant (open circle; <i>n</i> = 8) (F) HIV-Uninfected participant (X; <i>n</i> = 5). P values were calculated with repeated-measures one-way ANOVA, followed by Tukey’s multiple comparisons test.</p